Use of nitrite salts in chronic ischemia

ABSTRACT

Methods of treating a subject who has chronic tissue ischemia are disclosed. The methods can include administering to the subject a pharmaceutical composition comprising inorganic nitrite or a pharmaceutically acceptable salt thereof, for a time and in an amount sufficient to result in blood vessel growth in the ischemic tissue. The subject can be diagnosed as having a medical condition that results in persistent or recurring restriction of blood supply to a tissue, for example, peripheral artery disease, diabetes, atherosclerotic cardiovascular disease or defective wound healing. The methods can include the step of identifying a suitable subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Application No. 61/003,150, which was filed on Nov. 15,2007. For the purpose of any U.S. application that may claim the benefitof U.S. Provisional Application No. 61/003,150, the contents of thatearlier filed application are hereby incorporated by reference in theirentirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work described below was support by Grant No. HL080482, which wasawarded by the National Institutes of Health. The Government has certainrights in the invention.

TECHNICAL FIELD

This invention relates to methods and compositions useful for thetreatment of chronic tissue ischemia, and more particularly to methodsfor inducing new blood vessel growth in ischemic tissue.

BACKGROUND

Chronic tissue ischemia, i.e., persistent restriction of blood supply toa tissue, can impair tissue function and result in tissue and organdamage. Chronic tissue ischemia in critical organ systems or body parts,for example, heart, brain, kidneys, skin, limbs, or gastrointestinaltract, contributes significantly to human morbidity and mortality andthere is a continuing need for therapeutic strategies that restore bloodsupply to affected regions.

SUMMARY

The present invention is based, in part, on our discovery ofcompositions and methods that can be used to treat chronic tissueischemia, including chronic tissue ischemia associated with a disorder,trauma or a congenital defect. The chronic tissue ischemia encompassedby the methods of the invention can stem from any of a wide range ofmedical conditions that result in the persistent or recurringrestriction of blood supply to the tissue, for example, disorders suchas peripheral artery disease, type 1 or type 2 diabetes, atheroscleroticcardiovascular disease, intermittent claudication (which can manifest ascramping pain in the extremities due to inadequate blood supply),critical limb ischemic disease, stroke, myocardial infarction,inflammatory bowel disease, and peripheral neuropathy; traumaticinjuries such as wounds, burns, lacerations, contusions, hone fractures,infections, or surgical procedures; congenital malformations such ashernias, cardiac defects and gastrointestinal defects. Thus, chronictissue ischemia can occur in a variety of tissue types including, forexample, skeletal muscle, smooth muscle, cardiac muscle, neuronaltissue, skin, mesenchymal tissue, connective tissue, gastrointestinaltissue and bone.

Regardless of whether the methods are described with respect to aparticular medical condition or tissue type, the methods can be carriedout by administering to a subject (e.g., a human patient) in need oftreatment a pharmaceutically acceptable composition comprising inorganicnitrite or a pharmaceutically acceptable salt thereof. The inorganicnitrite or a pharmaceutically acceptable salt thereof can be formulatedin various ways and can include pharmaceutically acceptable carriers.For ease of reading, we will not repeat the phrase “or apharmaceutically acceptable salt thereof” on every occasion. It is to beunderstood that where inorganic nitrite can be used, a pharmaceuticallyacceptable salt of the compound may also be used.

Accordingly, the invention features physiologically acceptablecompositions of inorganic nitrite and methods by which the compositionscan be administered to a patient diagnosed as having, for example, achronic tissue ischemic disorder. These methods can include the steps ofa) identifying a subject (e.g., a human patient) who is experiencing oris likely to experience a chronic tissue ischemic disorder; and b)providing to the subject a composition including inorganic nitrite for atime and in an amount sufficient to stimulate blood vessel growth in theischemic tissue. The nitrite can result in the formation of a bloodvessel that did not exist prior to treatment or in an increase in thesize of existing vessels. The increase in size is due to formation ofnew tissue (e.g., new tissue added to the vessel wall); it is not theresult of simple vasodilation. Patients amenable to being treated withinorganic nitrite can also be treated with nitrate. We may use the terms“subject,” “individual” and “patient” interchangeably. While the presentmethods are certainly intended for application to human patients, theinvention is not so limited. Domesticated animals, including, forexample cats, dogs, horses, cows and other domesticated animals can alsobe treated.

The pharmaceutically acceptable compositions of the invention includeinorganic nitrite, e.g., a salt or ester of nitrous acid (HNO₂) or apharmaceutically acceptable salt thereof. As used herein,“pharmaceutically acceptable salts” refers to derivatives of thedisclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form. Examples ofpharmaceutically acceptable salts include, but arc not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts of the present inventioninclude the conventional non-toxic salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. Thepharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred.

Suitable pharmaceutically acceptable salts can include, for example,sodium nitrite, potassium nitrite, or calcium nitrite. The invention isnot so limited however and lists of exemplary salts are found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2(1977), each of which is incorporated herein by reference in itsentirety. It will also be understood that certain nitrite compounds ofthe present invention may exist in solvated, for example hydrated, aswell as unsolvated forms. Nitrite has the chemical formula NO_(2—) andmay exist as an ion in water. Sodium nitrite has the chemical formulaNaNO₂ and typically dissolves in water to form the sodium ion Na+ andthe nitrite ion NO_(2—). It will further be understood that the presentinvention encompasses all such solvated forms of the nitrite compounds.

The inorganic nitrite is administered for a time and in an amountsufficient to result in the growth of blood vessels in the ischemictissue. The new blood vessel growth may stem from any process thatresults in revascularization of the ischemic tissue, for example,angiogenesis, i.e., the budding of new capillary branches from existingblood vessels, or arteriogenesis, i.e., the growth of preexistingarteriolar connections into true collateral arteries, or a combinationof angiogenesis and arteriogenesis. New blood vessel grow may bemonitored over the course of treatment either directly, using, forexample imaging techniques such as contrast angiography, contrast pulsesequence (CPS) ultrasound imaging for high-resolution perfusion,biomarkers, or indirectly, i.e., by monitoring a clinical endpoint. Forexample, the nitrite may be administered until a symptom of chronicischemia, e.g., intermittent claudication, claudication during rest,neuropathy, or defective tissue wound healing, improves. The assessmentof clinical benefit may entail comparison of the ischemic tissue withthe corresponding non-ischemic tissue. Choice of specific clinicalendpoints may depend, in part, upon the nature of the underlying medicalcondition, e.g., cessation or amelioration of intermittent claudicationmay be useful for patients with peripheral artery disease or diabetes;healing of skin ulcers may be useful for patients with defective woundhealing, and relief from gastrointestinal pain, diarrhea andconstipation may be useful for patient suffering from bowel ischemia.

The amount of inorganic nitrite per dose can vary. For example, asubject can receive from about 0.05 μg/kg up to about 5000 μg/kg., e.g.,about 0.05, 1, 5, 10, 25, 50, 100, 200, 250, 300, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 2000, 2500, 3000,3500, 4000, 4500 or 5000 μg/kg. For example, a subject can receive up toor up to about 165 μg/kg, 16.5 μg/kg, or 8.25 μg/kg. Generally, weadminister nitrite in an amount such that the circulating concentrationdoes not exceed 0.6 μM (i.e., the nitrite is administered in a dosesufficient to produce a circulating concentration of nitrite in thesubject that does not exceed 0.6 μM). For example, the nitrite can beadministered in an amount such that the circulating concentration doesnot exceed 0.0005 μM, 0.001 μM, 0.002 μM, 0.003 μM, 0.004 μM, 0.005 μM,0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM,0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM or 0.6 μM.Thus, exemplary dosages can produce a circulating concentration ofnitrite in the subject of up to or up to about 0.03 μM, 0.003 μM, or0.0015 μM.

The frequency of treatment may also vary. The subject can be treated oneor more times per day (e.g., once, twice, three, four, five, or sixtimes per day) or every so-many hours (e.g., about every 2, 4, 6, 8, 12,or 24 hours). The time course of treatment may be of varying duration,e.g., for two, three, four, five, six, seven, eight, nine, ten or moredays. For example, the treatment can be twice a day for three days,twice a day for seven days, twice a day for ten days. Treatment cyclescan be repeated at intervals, for example weekly, bimonthly or monthly,which are separated by periods in which no treatment is given. Thetreatment can be a single treatment or can last as long as the life spanof the subject (e.g., many years).

The compositions can be administered to a subject in a variety of ways.For example, the compositions can he administered transdermally orinjected (infused) intravenously, subcutaneously, sublingually,intracranially, intramuscularly, intraperitoneally, or intrapulmonarily.Oral formulations arc also within the scope of the present invention.The treatment regime can vary depending upon various factors typicallyconsidered by one of ordinary skill in the art. These factors includethe route of administration, the nature of the formulation, the natureof the patient's illness, the subject's size, weight, surface area, age,gender, other drugs being administered to the patient, and the judgmentof the attending physician. The compositions can be administered alongwith or in addition to other treatments for chronic tissue ischemia,e.g., drug therapy, immunotherapy, or surgery (e.g., aspirin therapy,statin therapy, or antihypertensive therapy).

Disorders amenable to the methods of the invention can include anydisorder that presents with chronic ischemia. Conditions that result inchronic tissue ischemia due to a narrowing or blockage of an artery, forexample, include but are not limited to, for example, atherosclerosis,arteriosclerosis, acute coronary syndrome, coronary artery disease(CAD), stroke, bowel ischemia and peripheral artery diseases. Alsoencompassed by the invention is chronic tissue ischemia that stems froma wound, e.g. a traumatic injury or a surgical procedure.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A, 1B, 1C and 1D depict the results of an analysis demonstratingthat chronic sodium nitrite treatment restored ischemic hind limb bloodflow in an NO-dependent manner. effect of chronic sodium nitrite therapyon ischemic hind limb blood flow. FIG IA depicts the effect of variousdoses of chronic sodium nitrite therapy on ischemic hind-limb blood flowover time compared to PBS control. FIG. 1B depicts the effect of sodiumnitrite therapy plus 1 mg/kg carboxy PTIO treatment on ischemichind-limb blood flow. FIG. 1C depicts the effect of 165 μg/kg sodiumnitrite injection on ischemic limb blood flow. FIG. 1D depicts theeffect of PBS injection on ischemic limb blood flow. *, P<0.01 vs. PBScontrol at each time point; #, P<0.01 165 μg/kg sodium nitrite vs. 165μg/kg sodium nitrite plus cPTIO at each time point.

FIGS. 2A and 2B depict the results of an analysis demonstrating thatchronic sodium nitrite treatment did not alter non-ischemic hind-limbblood flow. FIG. 2A shows non-ischemic hind-limb blood flow in responseto intra-peritoneal injection of PBS. FIG. 2B depicts non-ischemichind-limb blood flow in response to intra-peritoneal injection of 165μg/kg sodium nitrite.

FIG. 3 depicts the results of an analysis of the effect of various dosesof chronic sodium nitrate treatment on ischemic hind-limb blood flow.

FIGS. 4A, 4B, 4C, 4D, 4E and 4F depict the results of an analysisdemonstrating that chronic sodium nitrite treatment increased ischemictissue vascular density in an NO-dependent manner. FIGS. 4A and 4Bdepict representative images of CD31 (red) and DAPI nuclear (blue)staining ischemic gastrocnemius muscle tissue at day 7 from sodiumnitrite-treated and sodium nitrate-treated animals, respectively. FIG.4C and FIG. 4D depict the vascular density of ischemic gastrocnemiusmuscle tissue at days 3 and 7 for 165 μg/kg sodium nitrite and nitratetreatments, respectively. FIG. 4E and FIG. 4F depict the vasculardensity of ischemic gastrocnemius muscle tissue at days 3 and 7 from 165μg/kg sodium nitrite plus carboxy PTIO. (Scale bar, 150 μm.).

FIGS. 5A, 5B and 5C depict the results of an analysis of vasculardensity measurements from high-dose sodium nitrite and non-ischemicnitrite tissues. FIG. 5A and FIG. 5B depict the vascular density ofischemic gastrocnemius muscle tissue for 3.3 mg/kg sodium nitrite andnitrate treatments at days 3 and 7, respectively. FIG. 5C depicts thevascular density measurements at day 7 of non-ischemic gastrocnemiusmuscle tissue from PBS-control and 165 μg/kg sodium nitrite-treatedmice.

FIGS. 6A, 6B, 6C and 6D depict the results of an experimentdemonstrating that chronic sodium nitrite treatment stimulatedendothelial cell proliferation in an NO-dependent manner. FIG. 6A andFIG. 6B depict representative images of Ki67 proliferation marker(green), CD31 (red), and DAPI nuclear (blue) staining from day 3ischemic gastrocnemius muscle tissue from PBS and 165 μg/kg sodiumnitrite-treated animals, respectively. FIG. 6C depicts the amount ofKi67 colocalization with DAPI nuclear staining between PBS and sodiumnitrite-treated tissues±cPTIO. FIG. 6D depicts the measurement of Ki67colocalization with CD31 staining between PBS and sodium nitrite-treatedtissues plus cPTIO. *, P<0.001 sodium nitrite vs. PBS or nitrite plusPTIO. (Scale bar, 150 μm.)

FIGS. 7A, 7B, 7C, 7D and 7E depict the results of an experimentdemonstrating that chronic sodium nitrite treatment altered blood andtissue nitrite levels. FIG. 7A depicts blood nitrite levels at days 3and 7 for PBS control and sodium nitrite (165 μg/kg). *, P<0.01treatments vs. PBS control blood nitrite levels. FIG. 7B and FIG. 7Cdepict tissue nitrite levels for PBS-treated control and sodiumnitrite-treated animals (165 μg/kg) at day 3 and 7, respectively. *,P<0.01 ischemic vs. nonischemic tissue nitrite levels. FIG. 7D and FIG.7E depict total cNOS protein expression normalized to β actin expressionfor PBS-treated and sodium nitrite-treated animals at day 3 and 7,respectively in ischemic and nonischemic hind-limbs.

FIGS. 8A, 8B, 8C and 8D depict the results of an experiment analyzingthe effect of chronic sodium nitrite treatment on tissue NO metabolitesand cGMP levels. FIG. 8A and FIG. 8B depict the amount of SNO+XNO levelsin nonischemic and ischemic tissues at day 3 and 7 for PBS control and165 μg/kg sodium nitrite, respectively. FIG. 8C and FIG. 8D report thepg/mg total protein of cGMP in nonischemic and ischemic tissues at day 3and 7 for PBS control and 165 μg/kg sodium nitrite, respectively.

FIGS. 9A, 9B, 9C, 9D and 9E depict the results of an experimentdemonstrating that chronic sodium nitrite treatment acutely increasedischemic tissue blood flow and stimulates arteriogenesis. FIG. 9A andFIG. 9B depict 165 μg/kg sodium nitrite-induced acute changes in bloodflow of chronically ischemic tissues at various time points with orwithout cPTIO, respectively. FIG. 9C depict the number of arterialbranches in PBS-treated control and sodium nitrite-treated animals. FIG.9D and FIG. 9E depict vascular casting of the arterial vasculature inischemic hind limbs of day 7 nitrite-treated and PBS-treated mice,respectively. *, P<0.01 vs. sodium nitrate.

FIG. 10 depicts the results of an experiment demonstrating that singlebolus I.P. injection of sodium nitrite did not restore ischemichind-limb blood flow.

FIG. 11 depicts the results of an experiment demonstrating that delayednitrite treatment restored ischemic hind-limb blood flow. *p<0.01 sodiumnitrite versus PBS at each respective time point.

FIG. 12 depicts the results of an experiment demonstrating thatcontinuous nitrite treatment enhanced wound healing. (*p<0.01 sodiumnitrite versus PBS at each respective time point.)

FIG. 13 depicts the results of an experiment demonstrating that sodiumnitrite restored Db/Db diabetic mouse ischemic hind-limb blood flow.(*p<0.01 sodium nitrite versus PBS at each respective time point.)

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

We further describe below the present methods for treatment of chronictissue ischemia. These methods can be applied to, and are expected tobenefit subjects having any of a variety of medical conditions that cangive rise to chronic tissue ischemia. The methods are based, inter alia,on the inventor's discovery that administration of inorganic nitrite ora pharmaceutical composition comprising inorganic nitrite to a subjecthaving chronic tissue ischemia results in the selective growth of newblood vessels in the ischemic tissue.

Compositions

The pharmaceutically acceptable compositions of the invention includeinorganic nitrite, e.g., a salt or ester of nitrous acid (HNO₂) or apharmaceutically acceptable salt thereof. The nitrite ion is NO2-. Moregenerally, a nitrite compound is either a salt or an ester of nitrousacid. Nitrite salts can include, without limitation, salts of alkalimetals, e.g., sodium, potassium; salts of alkaline earth metals, e.g.,calcium, magnesium, and barium: and salts of organic bases, e.g., aminebases and inorganic bases. Compounds of the invention also include allisotopes of atoms occurring in the intermediate or final compounds.Isotopes include those atoms having the same atomic number but differentmass numbers. For example, isotopes of hydrogen include tritium anddeuterium. The term, “compound,” as used herein with respect to anyinorganic nitrite or pharmaceutically acceptable salt thereof and ismeant to include all stereoisomers, geometric iosomers, tautomers, andisotopes of the structures depicted. All compounds, and pharmaceuticallyacceptable salts thereof, are also meant to include solvated or hydratedforms.

The compounds of the present invention can be prepared in a variety ofways known to one of ordinary skill in the art of chemical synthesis.The compounds of the present invention can be synthesized using themethods described below, together with synthetic methods known in theart of synthetic chemistry or variations thereon as appreciated by oneof ordinary skill in the art. Methods for preparing nitrite salts arewell known in the art and a wide range of precursors and nitrite saltsare readily available commercially. Nitrites of the alkali and alkalineearth metals can be synthesized by reacting a mixture of nitrogenmonoxide (NO) and nitrogen dioxide (NO₂) with a corresponding metalhydroxide solution, as well as through the thermal decomposition of thecorresponding nitrate. Other nitrites are available through thereduction of the corresponding nitrates.

The present compounds can be prepared from readily available startingmaterials using the following general methods and procedures. It will beappreciated that where typical or preferred process conditions (i.e.,reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are given, other process conditions can also be usedunless otherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one of ordinary skill in the art by routine optimizationprocedures.

Regardless of their original source or the manner in which they areobtained, the compounds of the invention can be formulated in accordancewith their use. For example, the compounds can be formulated withincompositions for application to cells in tissue culture or foradministration to a patient. When employed as pharmaceuticals, any ofthe present compounds can be administered in the form of pharmaceuticalcompositions. These compositions can be prepared in a manner well knownin the pharmaceutical art, and can be administered by a variety ofroutes, depending upon whether local or systemic treatment is desiredand upon the area to be treated. Administration may be topical(including ophthalmic and to mucous membranes including intranasal,vaginal and rectal delivery), pulmonary (e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal), ocular, oral orparenteral. Methods for ocular delivery can include topicaladministration (eye drops), subconjunctival, periocular or intravitrealinjection or introduction by balloon catheter or ophthalmic insertssurgically placed in the conjunctival sac. Parenteral administrationincludes intravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular administration. Parenteral administration can be inthe form of a single bolus dose, or may be, for example, by a continuousperfusion pump. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids, powders, and thelike. Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds described hereinin combination with one or more pharmaceutically acceptable carriers. Inmaking the compositions of the invention, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, sachet,paper, or other container. When the excipient serves as a diluent, itcan be a solid, semi-solid, or liquid material (e.g., normal saline),which acts as a vehicle, carrier or medium for the active ingredient.Thus, the compositions can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,syrups, aerosols (as a solid or in a liquid medium), ointmentscontaining, for example, up to 10% by weight of the active compound,soft and hard gelatin capsules, suppositories, sterile injectablesolutions, and sterile packaged powders. As is known in the art, thetype of diluent can vary depending upon the intended route ofadministration. The resulting compositions can include additionalagents, such as preservatives. The compounds may also be applied to asurface of a device (e.g., a catheter) or contained within a pump,patch, or other drug delivery device. The therapeutic agents of theinvention can be administered alone, or in a mixture, in the presence ofa pharmaceutically acceptable excipient or carrier (e.g., physiologicalsaline). The excipient or carrier is selected on the basis of the modeand route of administration. Suitable pharmaceutical carriers, as wellas pharmaceutical necessities for use in pharmaceutical formulations,are described in Remington's Pharmaceutical Sciences (E. W. Martin), awell-known reference text in this field, and in the USP/NF (UnitedStates Pharmacopeia and the National Formularly).

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thepharmaceutical compositions can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining, for example, from about 0.1 mg to about 50 mg, from about0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg, from about 0.1mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mgto about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg toabout 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg toabout 50 mg, from about 0.5 mg to about 30 mg, from about 0.5 mg toabout 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg toabout 5 mg; from about 1 mg from to about 50 mg, from about 1 mg toabout 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, fromabout 5 mg to about 20 mg, from about 5 mg to about 10 mg; from about 10mg to about 100 mg, from about 20 mg to about 200 mg, from about 30 mgto about 150 mg, from about 40 mg to about 100 mg, from about 50 mg toabout 100 mg of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient. For preparing solid compositions such as tablets, theprincipal active ingredient is mixed with a pharmaceutical excipient tofount a solid preformulation composition containing a homogeneousmixture of a compound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can he readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can he separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein and/or known in the art. In some embodiments, the compositionsare administered by the oral or nasal respiratory route for local orsystemic effect. Compositions in can be nebulized by use of inert gases.Nebulized solutions may be breathed directly from the nebulizing deviceor the nebulizing device can be attached to a face masks tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions can be administered orally or nasally fromdevices which deliver the formulation in an appropriate manner. Thecompositions administered to a patient can be in the form of one or moreof the pharmaceutical compositions described above. These compositionscan be sterilized by conventional sterilization techniques or may besterile filtered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between about 3 and 11, for example,between about 5 to 9, between 6 and 7, between 7 and 8. It will beunderstood that use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of pharmaceutical salts.

The proportion or concentration of a compound of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the compounds of the inventioncan be provided in an aqueous physiological buffer solution containingabout 0.1 to about 10% w/v of the compound for parenteraladministration.

Methods of Treatment

Chronic tissue ischemia is associated with a wide range of medicalconditions that result in partial, substantially complete or completereduction of blood flow to a body part or tissue comprising a body partand may be the result of disease, injury, or of an unknown cause, andmay be influenced by one's genetic constitution. Regardless of themedical condition leading to in chronic tissue ischemia, a patient whohas chronic tissue ischemia is a candidate for treatment with thepharmaceutically acceptable compositions comprising inorganic nitritedescribed herein. Treatment can completely or partially abolish some orall of the signs and symptoms of chronic tissue ischemia, decrease theseverity of the symptoms, delay their onset, or lessen the progressionor severity of subsequently developed symptoms.

New Blood Vessel Growth

As described further below, the compositions of the invention areadministered for a time and in an amount sufficient to result in thegrowth of new blood vessels in the ischemic tissue. We may use the terms“new blood vessel growth,” “new blood vessel formation” and “new bloodvessel development” interchangeably. New blood vessel growth refers allphases of the process of blood vessel formation, including the initialsignaling events, cellular recruitment of endothelial cells, theformation and enlargement of new vessels and connection of new vesselswith pre-existing vessels. The new blood vessel growth may stem from anyprocess that results in revascularization or neovascularization of theischemic tissue, for example, angiogenesis, or arteriogeneis, or acombination of angiogenesis and arteriogenesis. The term vasculogenesistypically is used to describe the embryonic development of blood vesselsfrom angioblasts. Angiogeneisis is generally understood to be apost-natal physiologic process required for would healing. Angiogenesisgenerally encompasses the formation of new capillaries or capillarybranches by sprouting, budding and intussusception from pre-existentcapillaries. Arteriogenesis i.e., the growth of preexisting arteriolarconnections into true collateral arteries, is generally understood toencompass the formation of mature arteries from pre-existentinterconnecting arterioles after an arterial occlusion. It shares somefeatures with angiogenesis, but the pathways leading to it can differ,as do the final results: arteriogenesis is potentially able to fullyreplace an occluded artery whereas angiogenesis typically cannot.Increasing the number of capillaries within the ischemic region cannotincrease blood flow when the limiting structure lies upstream of the newcapillaries; formation of new collateral vessels that divert blood flowaround the site of a blockage. In addition, the structures produced byangiogenesis and arteriogenesis differ in their cellular composition.Capillaries are tubes formed by endothelial cells which are supported byvascular pericytes. Arteries and veins are tubes that consist ofmultiple layers: the intima, which is composed of endothelial cells,pericytes, and a basement membrane; the media, which is composedprincipally of smooth muscle cells and their extracellular matrix; and,in the largest vessels, the adventitia, which is composed principally offibroblasts and their extracellular matrix.

Multiple signaling pathways contribute to new blood vessel growth. Atthe center of these pathways is hypoxia-inducinble factor 1 (HIF-1), aheterodimeric transcription factor composed of a constitutivelyexpressed HIF-1β subunit and an oxygen-regulated HIF-1α subunit. TheHIF-1α subunit is continually synthesized and degraded within adequatelyperfused cells; under hypoxic conditions, the degradation of HIF-1α isinhibited, leading to its accumulation and dimerization with HIF-1β, DNAbinding, recruitment of coactivators and transcriptional activation oftarget genes. The inbalence between oxygen supply and demand leads tohypoxia, which is a physiological stimulus that induces cells to produceangiogenic cytokines such as Vascular Endothelial Growth Factor (VEGF).These secreted proteins bind to their cognate receptors (VEGFRs) onendothelial cells and activate signal transduction pathways thatstimulate cells to undergo sprouting angiogenesis. VEGF causes a massivesignaling cascade in endothelial cells. Binding to VEGF receptor-2(VEGFR-2) starts a tyrosine kinase signaling cascade that stimulates theproduction of factors that variously stimulate vessel permeability(eNOS, producting NO), proliferation/survival (bFGF), migration(ICAMs/VCAMs/MMPs) and finally differentiation into mature bloodvessels. Other growth factors involved in new blood vessel formationinclude, for example, FGF which can promote proliferation anddifferentiation of endothelial cells, smooth muscle cells, andfibroblasts; VEGFR and NRP-1, which can integrate survival signals; Ang1and Tie2, which can stabilize vessels; PDGF (BB-homodimer) and PDGFR,which can recruit smooth muscle cells; TGF-β, endoglin and TGF-βreceptors, which can increase extracellular matrix production; MCP-1;Integrins αVβ3, αVβ5 and α5β1 which can bind matrix macromolecules andproteinases; VE-cadherin and CD31; ephrin, which can determine formationof arteries or veins; plasminogen activators, which can remodelextracellular matrix and release and activate growth factors;plasminogen activator inhibitor-1, which can stabilize blood vessels;NOS and COX-2; AC133, which can regulate angioblast differentiation; andId1/Id3 which can regulate endothalial transdifferentiation.

In addition to their ability to activate vascular endothelial cellswithin the ischemic tissue, certain angiogenic cytokines, such as VEGF,PLGF and stromal-derived growth factor 1 (SDF-1), stimulate themobilization and recruitment of a heterogeneous population of angiogeniccells from the bone marrow and other tissues to sites of angiogenesisand arteriogenesis. Cell types that can participate in these responsesarc known as circulating angiogenic cells and include endothelialprogenitor cells, myeloid, mesenchymal and hematopoietic progenitorcells.

Arteriogenesis seems to he triggered mainly by fluid shear stress, whichis induced by the altered blood flow conditions after an arterialocclusion. Arteriogenesis involves endothelial cell activation, basalmembrane degradation, leukocyte invasion, proliferation of vascularcells, neointima formation, remodeling of the extracellular matrix andcytokine participation. More specifically, mechanical stresses causeendothelial cells to produce chemical facilitators that begin theprocess of increasing diameter. An increase in shear stress causes anincrease in the number of monocyte chemoattractant protein-1 (MCP-1)molecules expressed on the surface of vessel walls as well as increasedlevels of TNF-α, bFGF, and MMP. MCP-1 increases the tendency ofmonocytes to attach to the cell wall. TNF-α provides an inflammatoryenvironment for the cells to develop while bFGF helps induce mitosis inthe endothelial cells. Finally, MMPs remodel the space around the arteryto provide room for the expansion of the new collateral artery.

Nitric oxide (NO) has been shown to positively regulate endothelial cellresponses in both angiogenesis and arteriogenesis. NO increases theexpression of various angiogenic factors, including VEGF, which,together with other mediators, increases NO levels via a positivefeedback mechanism. In addition to stimulating the growth of nascent andimmature blood vessels consisting of only fragile endothelial cells, NOrecruits perivascular mural cells, which stabilize vessels and allowthem to become fully functioning conduits. NO can also protect tissuesagainst ischemic damage by slowing cellular respiration. NO has beenshown to modulate several endothelial cell signaling pathways forexample, Erk1/2 and PKC.

The primary enzyme responsible for NO production in the cardiovascularsystem is endothelial nitric oxide synthase (eNOS) which is regulated bynumerous molecules and signaling pathways. Importantly, cNOS activity isalso largely responsible for systemic NO production as the amount ofenzyme expression is often directly proportional to NO metabolitelevels. NO readily diffuses across lipid bilayers and its biologicalfate is dictated predominately by reactions with metalloproteins andother free radical species; the classic example being activation of theheme enzyme soluble guanylate cyclase (sGC) which initiates a signalcascade leading to vessel dilation and platelet inhibition. In addition,NO may also be oxidized through various mechanisms resulting in theformation of nitrite which can be further oxidized to nitrate (NO3-).

Both nitrite and nitrate are involved in regulating production of NOfrom NOS independent pathways. Inorganic nitrite can undergo a oneelectron reduction back to NO through various mechanisms withoxygen-binding heme proteins (hemoglobin and myoglobin),deoxyhemoglobin, deoxymyoglobin, xanthine oxidoreductase, endothelialnitric oxide synthase, acidic disproportionation, and members of themitochondrial electron transport chain, e.g., mitochondrial hemeproteins all being potential electron donors. The ability of nitrite tobe reduced back to NO classifies it as a unique NO donor underbiological conditions, e.g., tissue ischemia, in which many of thesepotential reducing agents are active. NO interacts with severalintracellular targets to form various NO-containing species includingS-nitrosothiols, C— or N—S-nitroso compounds, and nitrosylheme adducts.Moreover, these nitroso-products may serve as a biological reservoir forNO, which can be liberated under certain conditions

Administration

The present methods for treating chronic tissue ischemia are carried outby administering an inorganic nitrite for a time and in an amountsufficient to result in the growth of new blood vessels in the ischemictissue.

The amount and frequency of administration of the compositions can varydepending on, for example, what is being administered, the state of thepatient, and the manner of administration. In therapeutic applications,compositions can be administered to a patient suffering from chronictissue ischemia in an amount sufficient to relieve or least partiallyrelieve the symptoms of chronic tissue ischemia and its complications.The dosage is likely to depend on such variables as the type and extentof progression of the chronic tissue ischemia, the severity of thechronic tissue ischemia, the age, weight and general condition of theparticular patient, the relative biological efficacy of the compositionselected, formulation of the excipient, the route of administration, andthe judgment of the attending clinician. Effective doses can beextrapolated from dose-response curves derived from in vitro or animalmodel test system. An effective dose is a dose that produces a desirableclinical outcome by, for example, improving a sign or symptom of chronictissue ischemia or slowing its progression.

The amount of inorganic nitrite per dose can vary. For example, asubject can receive from about 0.05 ug/kg to about 5000 ug/kg., e.g.,about 0.05, 1, 5, 10, 25, 50, 100, 200, 250, 300, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 2000, 2500, 3000,3500, 4000, 4500 or 5000 ug/kg. Generally, we administer nitrite in anamount such that the circulating concentration does not exceed 0.6 uM.,e.g., 0.0005 uM, 0.001 uM, 0.002 uM, 0.003 uM, 0.004 uM, 0.005 uM, 0.01uM, 0.02 uM, 0.03 uM, 0.04 uM, 0.05 uM, 0.1 uM, 0.15 uM, 0.2 uM, 0.25uM, 0.3 uM, 0.35 uM, 0.4 uM, 0.45 uM, 0.5 uM, 0.55 uM or 0.6 uM. Thus,exemplary dosages can include 8.25 ug/kg, 16.5 ug·kg or 165 ug/kg andexemplary circulating plasma concentrations can include 0.0015 uM, 0.003uM or 0.030 uM.

The frequency of treatment may also vary. The subject can be treated oneor more times per day (e.g., once, twice, three, four or more times) orevery so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours). Thetime course of treatment may be of varying duration, e.g., for two,three, four, five, six, seven, eight, nine, ten or more days. Forexample, the treatment can be twice a day for three days, twice a dayfor seven days, twice a day for ten days. Treatment cycles can berepeated at intervals, for example weekly, bimonthly or monthly, whichare separated by periods in which no treatment is given. The treatmentcan be a single treatment or can last as long as the life span of thesubject (e.g., many years).

Chronic Tissue Ischemia

Method of the invention arc applicable to any of a wide range of medicalconditions which have as their underlying feature a persistent reductionof or partial or complete blockage of blood flow to a tissue or organ.Thus, the methods are applicable to treatment of chronic tissue ischemiaassociated with a disorder, with a trauma or an environmental stress.The reduction in blood flow to a tissue can be, for example, the resultof a progressive blockage of an artery due to hardening and/or loss ofelasticity due to an atheromatous plaque or the presence of a clot.Reduction of blood flow to a tissue can also be the result of anenvironmental insult, for example, a traumatic injury or surgicalprocedure that interrupts the blood flow to a tissue or organ.Typically, the oxygen tension of a wound quickly and progressivelydecreases with the development of varying degrees of hypoxia throughoutthe wound region. Environmental conditions that induce hypoxia are alsowithin the scope of the invention.

Disorders encompassed by the invention include, for example,cardiovascular disease, peripheral artery disease, arteriosclerosis,atherosclerotic cardiovascular disease, myocardial infarction, criticallimb ischemic disease, stroke, acute coronary syndrome, intermittentclaudication, diabetes, including type 1 and type 2 diabetes, skinulcers, peripheral neuropathy, inflammatory bowel disease, ulcerativecolitis, Crohn's disease, intestinal ischemia, and chronic mesentericischemia.

The methods of the invention are also applicable to chronic tissueischemia associated with a trauma, for example, a traumatic injury suchas a wound, laceration, burn, contusion, bone fracture or chronicinfection. Also encompassed by the invention are tissue injuriessustained as part of any surgical procedure, for example,endarterectomy. Procedures involving tissue or organ transplantation arewithin the scope of the invention. Examples include vascular bypassgrafts, heart, liver, lung, pancreatic islet cell transplantation aswell as transplantation of tissues generated ex vivo for implantation ina host. The methods of the invention are also useful for treating achronic ischemic condition brought about by exposure to an environmentalinsult, for example, chronic exposure to hypoxic conditions e.g., highaltitude, or sustained aerobic exertion.

The methods provided herein are applicable to any of a wide range oftissue types including, for example, muscle, smooth muscle, skeletalmuscle, cardiac muscle, neuronal tissue, skin, mesechymal tissue,connective tissue, gastrointestinal tissue or bone. Soft tissue, such asepithelial tissue, e.g., simple squamous epithelia, stratified squamousepithelia, cuboidal epithelia, or columnar epithelia, loose connectivetissue (also known as areolar connective tissue), fibrous connectivetissue, such as tendons, which attach muscles to bone, and ligaments,which join bones together at the joints.

The methods of the invention can include the steps of identifying asubject (e.g., a human patient) who is experiencing or is likely toexperience chronic tissue ischemia. Since chronic tissue ischemia canresult from a wide range of medical conditions all of which have astheir underlying feature a persistent reduction of or partial orcomplete blockage of blood flow to a tissue, the specific signs andsymptoms will vary depending upon factor or factors responsible for thereduction of blood flow.

Thus, for example symptoms of chronic tissue ischemia in peripheralartery disease (PAD), a form of peripheral vascular disease in whichthere is partial or total blockage of an artery, usually due toatherosclerosis in a vessel or vessels leading to a leg or arm, caninclude intermittent claudication, that is, fatigue, cramping, and painin the hip, buttock, thigh, knee, shin, or upper foot during exertionthat goes away with rest, claudication during rest, numbness, tingling,or coldness in the lower legs or feet, neuropathy, or defective tissuewound healing. PAD in the lower limb is often associated with diabetes,particularly type 2 diabetes. Arm artery disease is usually not due toatherosclerosis but to other conditions such as an autoimmune disease, ablood clot, radiation therapy, Raynaud's disease, repetitive motion, andtrauma. Common symptoms when the aim is in motion include discomfort,heaviness, tiredness, cramping and finger pain. PAD can be diagnosed byperforming one or more diagnostic tests including, for example, an anklebrachial index (ABI) test, angiography, ultrasound, or MRI analysis.

Myocardial ischemia can have few or no symptoms, although typically, itis associated with a symptoms such as angina, pain, fatigue elevatedblood pressure. Diagnostic tests for myocardial ischemia include:angiography, resting, exercise, or ambulatory electrocardiograms;scintigraphic studies (radioactive heart scans); echocardiography;coronary angiography; and, rarely, positron emission tomography.

The method of the invention can also be used in conjunction with otherremedies known in the art that are used to treat chronic tissue ischemiaincluding, drug therapy, surgery, anti-inflammatory agents, antibodies,exercise, or lifestyle changes. The choice of specific treatment mayvary and will depend upon the severity of the chronic tissue ischemia,the subject's general health and the judgment of the attendingclinician. The present compositions can also be formulated incombination with one or more additional active ingredients, which caninclude any pharmaceutical agent such antihypertensives, anti-diabeticagents, statins, anti-platelet agents (clopidogrel and cilostazol),antibodies, immune suppressants, anti-inflammatory agents, antibiotics,chemotherapeutics, and the like.

EXAMPLES Example 1 Materials and Methods

Animals and Reagents. Unless otherwise stated, male wild type (C57BL/6J)mice weighing 20-25 gm and age 2-3 months were used. The mice were bredand housed at the Association for Assessment and Accreditation ofLaboratory Animal Care, International-accredited LSUHSC-Shreveportanimal resource facility and maintained according to the NationalResearch Council's Guide for Care and Use of Laboratory Animals. Allexperimental protocols were approved by the LSU Institutional AnimalCare and Use Committee. Sodium nitrite, sodium nitrate, phosphatebuffered saline (PBS), and all other chemicals were purchased from SigmaChemical (St. Louis, Mo.). Hind-Limb Ischemia Model. Hind limb ischemiawas induced by ligating the left common femoral artery proximal toorigin of profunda femoris artery according to Senthilkumar, A., Smith,R. D., Khitha, J., Arora, N., Veerareddy, S., Langston, W., Chidlow, J.H., Jr., Barlow, S. C., Teng, X., Patel, R. P., et al. 2007.Arterioscler Thromb Vase Biol 27:1947-1954. Mice were anesthetized withintraperitoneal injection of ketamine (100 mg/kg) and xylazine (8mg/kg); surgery was performed unused aseptic conditions. The commonfemoral vein and femoral nerve were dissected away from the artery. Twoligatures were placed in the common femoral artery proximal to theprofunda femoris artery and then transected between the two ligations.The incision was then closed and the ligation was immediately verifiedby laser Doppler measurement of tissue blood flow.

Vascular casting. Hind limb vascular casting of ischemic limbs wasperformed using Microfil silicone injection MV120. Before vascularcasting, papaverine (5 ng/ml) and adenosine (1 mg/ml) were givenintravenously to increase perfusion of the casting resin. Briefly, theabdominal aorta proximal to the femoral artery bifurcation was ligatedwith 5.0 silk suture material and cannulated caudal to the ligationusing PESO tubing held in place with a silk ligature. 400 μl of Microfilwas infused into the aortic catheter and allowed to set in situ for 16hours at 4° C. Hind limb muscle tissue was cleared by incubating tissuesin graded glycerol solutions of 40%, 60%, 80%, and 100% for 24 hourseach. Vascular casts were subsequently photographed using astereomicroscope.

Laser Doppler measurements of tissue blood flow. The Vasamedics LaserfloBPM2 deep tissue penetrating laser doppler device was used to measurehind limb blood flow. The tip of laser probe was placed with stablepositioning using a probe stand over the medial calf muscle of mice.Readings were recorded in ml of blood flow per 100 g tissue per minutefrom non-ischemic and ischemic limbs. Daily blood flow measurements weretaken before the first nitrite or nitrate injection of a 24 hour periodin order to obtain representative steady state changes in perfusion.Percent change in tissue blood flow was determined by dividing ischemiclimb blood flow by non-ischemic limb blood flow and multiplied by 100.In a separate series of experiments, blood flow was also measured within30 seconds after nitrite or nitrate administration to assess acutechanges in blood flow.

Vascular density measurement. Determination of the vascular density ofmuscle tissue was performed as described in Senthilkumar, A., Smith, R.D., Khitha, J., Arora, N., Veerareddy, S., Langston, W., Chidlow, J. H.,Jr., Barlow, S. C., Teng, X., Patel, R. P., et al, 2007. ArteriosclerThromb Vase Biol 27:1947-1954. Briefly, ischemic (left) and non-ischemic(right) tissues were dissected and embedded in OCT freezing medium andfrozen and 3 micron sections cut. Slides were fixed at −20° C. in 95%ethanol/5% glacial acetic acid for one hour. Slides were blockedovernight with 5% horse serum in PBS at 4° C. Anti-CD31 (PECAM-1) wasadded at 1:200 dilution (in PBS with 0.05% horse serum) and incubated at37° C. for one hour. Slides were washed 3 times with 1% horse serum/PBSand Cy3 conjugated anti-rat secondary antibody was added at 1:250dilution (in PBS with 0.05% horse serum) and incubated at roomtemperature for one hour. Slides were washed and mounted usingVectashield DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride)nuclear counterstain. At least 4 fields were acquired per section with 4sections stained per muscle specimen. Pictures were taken with aHamamatsu digital camera using a Nikon TE-2000 epifluorescent microscope(Nikon Corporation, Japan) with TRITC and DAPI illumination at 200×magnification for CD31 and DAPI staining, respectively. Simple PCIsoftware version 6.5 (Compix Inc, Sewickly, Pa.) was used to quantitatethe surface area of CD31 and DAPI staining per section. Vascular densitywas measured as the ratio between CD31 pixel density divided by DAPIpixel density. Image acquisition and vascular density measurements wereaccumulated, analyzed, and calculated in a double blinded fashion beforeidentity of the data were used for statistical analysis and graphgeneration.

Measurement of Nitrite, NO Metabolite, Tissue cGMP, and eNOS ProteinLevels. Nitrite and tissue NO metabolite levels tissue (nitrosothiol, C-or N-nitroso compounds, or iron-nitrosyl proteins which are collectivelyreferred to SNO+XNO) whether were measured using chemiluminescencetechniques according to Lang, J. D., Jr., Teng, X., Chumley, P.,Crawford, J. H., Isbell, T. S., Chacko, B. K., Liu, Y., Jhala, N.,Crowe, D. R., Smith, A. B., et al. 2007. J Clin Invest. 117:2583-2591.Gastrocnemius muscle tissue cGMP levels were determined using the cGMPELISA from Cayman Chemical (Ann Arbor, Mich.) according to themanufacturer's directions. Specimens were collected within 2 min afterinjection of either PBS or sodium nitrite. Total eNOS protein levels inhind-limb tissue were determined by Western blot analysis.

Statistical analysis. Blood flow, vascular density, endothelial cellproliferation, tissue nitrate and NO metabolites, and cGMP data wereanalyzed using Students T-test (unpaired) between sodium nitrite orsodium nitrate vs. PBS control groups with a minimum of P<0.05 necessaryfor significance. Blood nitrite measurements and acute changes in bloodflow from experimental groups were compared against PBS controls and day0 time points, respectively, using one-way ANOVA with Bonferroni'spost-test with a minimum of p<0.05 necessary for significance.Statistics were done with GraphPad Prism 4.0 software. The number ofmice used per reported experiment is designated in the Examples below.

Example 2 Chronic Sodium Nitrite Therapy Increased Ischemic Tissue BloodFlow

We examined the effect of a range of sodium nitrite doses on ischemictissue blood flow in the mouse hind-limb ischemia model described inExample 1. Sodium nitrite was administered via intraperitoneal injectiontwice a day in a dose range of 8.25-3,300 μg/kg; 15 mice were used pertreatment group. Blood flow was measured according the laser Dopplermethod of Example 1. Changes in ischemic tissue perfusion weredetermined by measuring ischemic limb blood flow which was divided bythe respective contra-lateral non-ischemic limb blood flow to calculatea percent change for each animal. As shown in FIG. 1A, nitrite doses of8.25, 16.5, 165, and 3,300 μg/kg all increased the percentage blood flowin ischemic hind limbs by day 3 post ligation which progressivelyincreased by day 7. The 165 μg/kg sodium nitrite dose revealed optimaltemporal efficacy compared to higher and lower doses. We next examinedwhether sodium nitrite increased ischemic tissue blood flow through anitric oxide (NO) dependent mechanism. As shown in FIG. 1B, the NOscavenger, carboxy PTIO(2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) at adose of 1 mg/kg daily, significantly attenuated the ability of sodiumnitrite to restore ischemic limb blood flow. Injection of 165 μg/kgsodium nitrite enhanced blood flow in hind limbs that were ischemic for24 h, as shown in FIG. 1C; no changes in bloof flow were observed inhind limbs that were ischemic for 24 h that were injected with PBSinjection (FIG. 1D). Importantly, neither PBS nor sodium nitrite alteredhind limb blood flow in the nonischemic limbs as shown in FIGS. 2A and2B, respectively; 10 mice were used per treatment group. As shown inFIG. 3, when sodium nitrate was used instead of sodium nitrite, thelowest dose of sodium nitrate (8.25 μg/kg) augmented ischemic limb bloodflow by days 5 and 7.

Example 3 Chronic Sodium Nitrite Therapy Selectively IncreasedIschemia-Induced Angiogenesis

We next examined the effect of chronic sodium nitrite therapy onischemic tissue vascular density. Vascular density was measuredaccording to the method of Example 1. FIGS. 4A and 4B illustrateendothelial cell CD31 staining in red with DAPI nuclear counterstainingin blue of ischemic gastrocnemicus muscle at day 7 from animals treatedwith sodium nitrate or sodium nitrite (165 μg/kg), respectively.Endothelial staining of CD31 was much more abundant in ischemic muscletissue in mice receiving sodium nitrite as compared to PBS or sodiumnitrate controls. Quantitative analysis of vascular density revealedthat sodium nitrite (165 μg/kg) therapy significantly increased vasculardensity at days 3 and 7 compared to PBS or sodium nitrate controltreatments (FIGS. 4C and 4D); 10 mice were used per treatment group.Consistent with changes in tissue blood flow, cPTIO cotreatmentprevented nitrite augmentation of angiogenesis in the ischemic hindlimbs at days 3 and 7 (FIGS. 4E and 4F). High-dose sodium nitrite (3,300μg/kg) was less potent in augmenting ischemic tissue vascular densitycompared to low-dose (165 μg/kg) nitrite, consistent with observationsof percentage blood flow changes in ischemic tissues (FIGS. 5A and 5B).Importantly, sodium nitrite did not significantly alter nonischemictissue vascular density, highlighting the site-specific activity ofchronic nitrite therapy (FIG. 5C); 10 mice were used in each group.

Example 4 Chronic Sodium Nitrite Therapy Increased Ischemic EndothelialCell Proliferation

The effect of sodium nitrite on endothelial cell proliferation inischemic gastrocnemicus tissues was analyzed in the hind-limb ischemiamodel of Example 1. FIGS. 6A and 6B illustrate the amount of endothelialcell proliferation as determined by Ki67 staining (green) of ischemictissues along with endothelial cell CD31 labeling (red) and DAPI nuclearstaining (blue) from day 3 sodium nitrate or nitrite (165 μg/kg)treatments, respectively. FIG. 6C quantifies the amount of Ki67colocalization with DAPI nuclear counter stain at day 3 for sodiumnitrite or nitrate, and demonstrated that sodium nitrite significantlyincreased Ki67 nuclear localization in ischemic but not non-ischemictissues and that the increase in KI67 nuclear localization was blockedby carboxy PTIO. Moreover, sodium nitrite significantly increased Ki67to CD31 colocalization in ischemic tissues and this increase incolocalization was blocked by cPTIO (FIG. 6D); 10 mice were used pertreatment group.

Example 5 Effect of Chronic Nitrite Administration on Blood and TissueNitrite Levels

Analysis of blood and tissue nitrite levels in nitrate-treated andcontrol animals was performed according to the method in Example 1. Asshown in FIG. 7A blood levels of nitrite significantly decreased overtime in the 165-μg/kg sodium nitrite therapy and were minimally alteredwith PBS treatment. FIGS. 7B and 7C show tissue nitrite levels at day 3and day 7, respectively, of nitrite treatment; 10 mice were used pertreatment group. Nitrite therapy significantly increased levels oftissue nitrite in ischemic but not nonischemic tissue nitrite at day 3(FIG. 7B). At day 7 tissue nitrite levels did not differ significantlybetween nitrite-treated animals and PBS treated control animals (FIG.7C). Together, these data demonstrated that chronic sodium nitritetherapy resulted in preferential early tissue accumulation of nitrite inischemic versus nonischemic tissues. The decreased blood nitrite levelsin response to chronic sodium nitrite therapy prompted us to examinetotal eNOS protein levels in hind-limb muscle tissue from PBS or 165μg/kg sodium nitrite therapy. FIG. 7D shows that at day 3, chronicsodium nitrite therapy significantly decreased eNOS protein expressionin ischemic hind-limb tissue without altering expression in nonischemichind-limb tissue. At day 7, as shown in FIG. 7E, sodium nitrite reducedof eNOS protein expression in nonischemic tissue with no effect onischemic tissue; 3 mice were used per experimental group for theexperiment show in FIG. 7. These data suggested that the decrease inblood nitrite levels could be due to decreased eNOS protein expressionin response to chronic sodium nitrite therapy.

Example 6 NO Tissue Metabolites and Tissue cGMP During Chronic SodiumNitrite Therapy

The effect of nitrite treatment on levels of the NO metabolites, SNO andXNO was measure according to the method in Example 1. Twenty mice wereused per treatment group. Tissue SNO+XNO levels for 165 μg/kg sodiumnitrite-treated animals or PBS-treated controls at day 3 and 7 are shownin FIGS. 8A and 8B, respectively Neither sodium nitrite (165 μg/kg) norPBS significantly altered tissue SNO+XNO levels at day 3. However,nitrite therapy significantly increased SNO+XNO levels in ischemictissue at day 7 compared to PBS. These data revealed a delayed effect ofnitrite therapy on tissue nitrosothiol, C-/N-nitroso compounds, andiron-nitrosyl proteins and demonstrated preferential production of NOcontaining intermediates in ischemic tissues.

We also examined whether chronic sodium nitrite therapy selectivelyincreased cGMP levels in nitrite-treated animals. As shown in FIGS. 8Cand 8D, neither 165 μg/kg sodium nitrite nor PBS significantly alteredtissue cGMP levels at either day 3 or 7, respectively.

Example 7 Chronic Sodium Nitrite Therapy Augmented Arteriogenesis andAcute Changes in Ischemic Tissue Blood Flow

Stimulation of angiogenesis alone during chronic ischemia is generallyinsufficient for restoring tissue perfusion. Increased arteriogenesisthrough the recruitment and differentiation of smaller arterioles isimportant to supply newly formed microvasculature. Moreover, an acuteincrease in vascular shear stress due to increased blood flow is acritical mediator of arteriogenesis. We examined the effect of 165 μg/kgsodium nitrite on acute changes (30 sec) in ischemic and nonischemictissue perfusion according to the method in Example 1. Ten mice wereused per treatment group. As shown in FIG. 9A, 165 μg/kg sodium nitritesignificantly enhanced ischemic tissue blood flow by 92.3±18% within 30sec of administration at day 1 after ligation. The duration of increasedblood flow after sodium nitrite injection persisted >10 min (data notshown). The ability of nitrite to induce large increases in acute bloodflow was inversely proportional to the duration of tissue ischemia,because animals receiving chronic nitrite therapy showed a lesser, yetstill significant, increase in acute blood flow change at days 3 and 7.This observation could be because significant angiogenic activity hadoccurred, thereby diminishing the degree of tissue ischemia. The sodiumnitrite-dependent changes in acute tissue blood flow involved NO,because, as shown in FIG. 9B, cPTIO significantly blunted this responseat early time points.

To further evaluate arteriogenesis activity, we performed hind-limbligations distal to the profunda femoris and proximal to the knee tomore easily distinguish changes in the arterial supply. As shown in FIG.9C, the number of arterial branch points was significantly greater intissues from sodium nitrite-treated animals that in correspondingtissues from PBS-treated control animals. Arterial casts were madeaccording to the method in Example 1. FIG. 9D shows a representativeexample of an arterial cast from a sodium nitrite-treated ischemic limbat day 7; FIG. 9E shows an arterial cast of a PBS-treated ischemic limbat day 7. Numerous collateral vessels could be observed throughout thetissue in response to nitrite therapy, indicating enhancedarteriogenesis. Conversely, PBS treatment did not enhance arterialperfusion, and minimal collateral vessels are observed. Together, thesedata demonstrate that chronic nitrite therapy augmented arteriogenesisactivity in ischemic tissue.

Example 8 Effect of Single Bolus I.P. Injection of Sodium Nitrite onIschemic Hind Limb Blood Flow

Permanent femoral artery ligation was performed on the left hind limb ofC57BL/6J mice (n=3 per treatment group) according to the method inExample 1. A single bolus injection of either PBS or sodium nitrite (165μg/kg) was given I.P. 45 minutes after completing the ligation. Bloodflow recovery was monitored every other day over a 7 day period toevaluate the efficacy of single bolus injection therapy. As shown inFIG. 10, a single bolus I.P. injection of sodium nitrite did not restoreischemic hind limb blood flow. “Day 0 Pre” on the x-axis refers to bloodflow on the day of ligation before the ligation was performed; “Day 0Post” on the x-axis refers to blood flow on the day of ligation afterthe ligation was performed.

Example 9 Effect of Delayed Sodium Nitrite Therapy on Ischemic Hind LimbBlood Flow

We also evaluated the effect of delaying chronic sodium nitritetreatment on restoration of hind limb blood flow. Permanent femoralartery ligation was performed on the left hind limb of C57BL/6J mice(n=6 per treatment group) as described in Example 1. PBS or sodiumnitrite (165 μg/kg) I.P. injections (b.i.d.) were started the morning ofday 5 post-ligation and continued daily throughout the remainder of thestudy. Blood flow recovery was monitored throughout the study using deeptissue penetrating laser Doppler as described in Example 1. As shown inFIG. 11, delayed nitrite therapy restored ischemic hind limb blood flow.“Day 0 Pre” on the x-axis refers to blood flow on the day of ligationbefore the ligation was performed; “Day 0 Post” on the x-axis refers toblood flow on the day of ligation after the ligation was performed.

Example 10 Sodium Nitrite Treatment Restored Ischemic Hind-Limb BloodFlow in Diabetic Mice

We evaluated the effect of sodium nitrite treatment on ischemic hindlimb blood flow in a diabetic mouse model. Permanent femoral arteryligation was performed on the left hind limb of Db/Db diabetic mice (n=5per treatment group) according to the methods in Examples 1 and 2,except that the mice were anesthetized with 2% inhaled isofluorane. PBSor sodium nitrite (165 μg/kg) I.P. injections (b.i.d.) were startedpost-ligation and continued daily throughout the remainder of the study.Blood flow recovery was monitored throughout the study using deep tissuepenetrating laser Doppler according to the method in Example 1. As shownin FIG. 13, sodium nitrite treatment restored ischemic hind-limb bloodflow in diabetic mice. Blood flow in the ischemic-hind limbs of thenitrite-treated animals was significantly greater than that of thePBS-treated control animals at all time points analyzed and approachedpreligation levels by the end of the study.

Example 11 Continuous Sodium Nitrite Therapy Enhanced Excision WoundHealing

Full thickness excisional wounds were created using a 4 mm punch biopsydevice. C57BL/6J mice were anesthetized according to the method inExample 1. Wounds were created by pinching the loose skin and punching ahalf circle biopsy to obtain a uniform 4 mm diameter wound (n=4 pertreatment group). PBS or sodium nitrite (165 μg/kg) I.P. injections(b.i.d.) were began immediately after wounding and continued dailythroughout the remainder of the study. Wound diameter was measured usingdigital calipers and plotted as the percent wound surface area remainingat each successive day. As shown in FIG. 12, continuous sodium nitritetherapy enhanced excision wound healing. The percent wound surfaceremaining was significantly less in the sodium nitrite-treated animalsthan in the PBS-treated control animals for the first six days of thestudy, and remained, lower for the remaining four days.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-29. (canceled)
 30. A method of treating peripheral neuropathy in ahuman subject, the method comprising: orally administering to thesubject about 5 mg to about 50 mg of sodium nitrite one to six times perday for at least ten days, thereby treating the peripheral neuropathy.31. The method of claim 31, wherein the sodium nitrite is administereduntil a symptom of peripheral neuropathy in the subject improves. 32.The method of claim 31, wherein the sodium nitrite is administered to acirculating nitrite concentration in the subject of about 0.01 μM toabout 0.6 μM.
 33. The method of claim 31, wherein the method stimulatesblood vessel growth in ischemic tissue but not in non-ischemic tissue.34. The method of claim 33, wherein the ischemic tissue comprisesskeletal muscle, smooth muscle, cardiac muscle, neuronal tissue, skin,mesenchymal tissue, connective tissue, gastrointestinal tissue or bone.35. The method of claim 34, wherein administration of said sodiumnitrite alters nitrite levels in the blood.
 36. The method of claim 34,wherein administration of said sodium nitrite raises nitrite levels inthe ischemic tissue.
 37. The method of claim 1, wherein the sodiumnitrite is administered two times per day.
 38. A method of treating painassociated with chronic ischemia in a human subject, the methodcomprising: orally administering to the subject about 5 mg to about 50mg of sodium nitrite one or six times per day for at least ten days,thereby treating the pain.
 39. The method of claim 38, wherein thesodium nitrite is administered until the pain associated with chronicischemia improves.
 40. The method of claim 38, wherein the sodiumnitrite is administered to a circulating nitrite concentration in thesubject of about 0.01 μM to about 0.6 μM.
 41. The method of claim 38,wherein the method stimulates blood vessel growth in ischemic tissue butnot in non-ischemic tissue.
 42. The method of claim 41, wherein theischemic tissue comprises skeletal muscle, smooth muscle, cardiacmuscle, neuronal tissue, skin, mesenchymal tissue, connective tissue,gastrointestinal tissue or bone.
 43. The method of claim 42, whereinadministration of said sodium nitrite alters nitrite levels in theblood.
 44. The method of claim 41, wherein administration of said sodiumnitrite raises nitrite levels in the ischemic tissue.
 45. The method ofclaim 38, wherein the sodium nitrite is administered two times per day.46. A method of promoting angiogenesis or arteriogenesis in ischemictissue in a human subject having a chronic ischemic disorder, the methodcomprising: orally administering to the subject about 5 mg to about 50mg of sodium nitrite one or six times per day, thereby promotingangiogenesis or arteriogenesis in the ischemic tissue.
 47. The method ofclaim 46, wherein the sodium nitrite is administered until a symptom ofchronic ischemia in the subject improves.
 48. The method of claim 46,wherein the sodium nitrite is administered to a circulating nitriteconcentration in the subject of about 0.01 μM to about 0.6 μM.
 49. Themethod of claim 46, wherein administration of said sodium nitrite raisesnitrite levels in the ischemic tissue.
 50. The method of claim 46,wherein the chronic ischemic disorder is peripheral artery disease,diabetes, atherosclerotic cardiovascular disease, intermittentclaudication, critical limb ischemic disease, defective wound healing,stroke, myocardial infarction, inflammatory bowel disease, a bonefracture, or a bone infection.